We have obtained and analyzed cDNA clones from several genes of various rotavirus strains. Single or double stranded RNAs were used for cDNA synthesis by reverse transcriptase after either polyadenylation and oligo (dT) priming or direct priming with synthetic oligonucleotides corresponding to the known end sequences of the genes. Double stranded cDNA molecules were inserted into plasmid vectors by either d.D./d.G. tailing or direct blunt end ligation. In many cases the RNA templates used for reverse transcription consisted of individually isolated genes (by preparative PAGE); otherwise identification of the gene segment present in each clone was established by Northern blot, hybridization or colony hybridization with cDNA probes from clones of known origin. Clones are now available that include copies of the VP7 gene of the animal rotavirus strains NCDV and UK (bovine), OSU (porcine), and RRV (simian) and the human rotavirus strains Wa, DS-1, P, M37 and ST3. Also, VP3 gene clones from RRV and NCDV have been obtained. Direct dideoxy sequencing of either mRNAs or cloned cDNAs has been performed for the VP7 gene of a number of strains by using sequence-specific oligonucleotide synthetic primers. In addition to the OSU and NCDV VP7 glycoprotein gene sequences, the complete sequence of the RRV VP7 glycoprotein gene has been determined as well as most of the corresponding sequence of the human strains D (serotype 1), DS-1 (serotype 2), P (serotype 3) and ST3 and VA70 (both serotype 4).